Unconjugated hyperbilirubinemia in the newborn may cause encephalopathy with resultant minimal to severe residual brain damage or death. Exchange transfusion provides effective preventive therapy if instituted before bilirubin toxicity is irreversible. In an effort to improve laboratory evaluation of jaundiced infants at risk of kernicterus, we have recently developed a sensitive automated enzymatic assay for unbound bilirubin in icteric serum which permits both determination of the unbound bilirubin concentration and rapid characterization of bilirubin-albumin binding (binding affinity, binding capacity). We propose to study the pathogenesis of bilirubin toxicity and evaluate potential clinical usefulness of the peroxidase assay for managing jaundiced infants. Specifically, we shall (1) assess potential errors produced by serum dilution, conjugated bilirubin, and agents affecting the reaction velocity, (2) attempt to correlate unbound bilirubin concentration with tissue bilirubin uptake and toxicity in vitro, and (3) determine day-to-day variations of binding in sick jaundiced infants and compare the peroxidase assay with other binding tests used in managing these patients.